脑红蛋白改善AD双转基因（APPswe/PS1dE9）小鼠认知功能及其机制研究

Neuroglobin improves cognitive function of AD double transgenic（APPswe/PS1dE9） mice and its mechanisms

摘要:

目的：观察过表达脑红蛋白（neuroglobin，Ngb）对阿尔茨海默病（Alzheimer’s disease，AD）双转基因（APPswe/PS1dE9）小鼠认知功能的影响，并探讨其机制。方法：成功构建过表达Ngb质粒pNgb和对应的空质粒pCDNA3.1，对AD双转基因小鼠进行侧脑室注射，使其过表达Ngb。采用Morris水迷宫评价过表达Ngb对AD小鼠学习记忆能力和空间探索能力的影响；采用免疫组织化学法检测其对β淀粉样蛋白-42（amyloid β-protein 42，Aβ42）生成的影响；半胱氨酸蛋白酶Caspase-3和Caspase-9酶活性检测试剂盒检测酶活性变化；Western blot检测海马组织中磷酸化蛋白激酶B p-Akt（ser473）、总Akt、Caspase-3和Caspase-9表达。结果：过表达Ngb提高了AD转基因小鼠的学习记忆和空间探索能力（P<0.05）；明显降低了转基因小鼠海马组织中Caspase-3和Caspase-9酶活性及Aβ42生成（P<0.05）；Western blot显示过表达Ngb能抑制脑组织中Caspase-3和Caspase-9蛋白表达（P<0.05），而且还能促进p-Akt（ser473）表达（P<0.05）；应用磷脂酰肌醇-3激酶（phosphatidylinositol-3 kinase，PI3K）抑制剂LY294002后，Caspase-3和Caspase-9活性明显提高（P<0.05）。结论：过表达Ngb对AD双转基因（APPswe/PS1dE9）小鼠有明显的神经保护作用，其机制与Ngb减少Aβ42生成及激活PI3K/Akt信号通路进而抑制Caspase依赖的细胞凋亡途径有关。

Abstract:

Objective：To observe the neuroprotective effect of overexpression of neuroglobin（Ngb） on the cognitive function of Alzheimer’s disease（AD） double transgenic（APPswe/PS1dE9） mice，and to explore its mechanisms. Methods：The Ngb plasmid pNgb and empty vector pCDNA3.1 were successfully constructed and injected into the lateral ventricle of AD double transgenic（APPswe/PS1dE9） mice，which both led to overexpression of Ngb. Morris water maze was used to evaluate the effect of overexpression of Ngb on learning and memory ability and spatial exploration ability of AD mice；immunohistochemistry was used to detect the production of amyloid β-protein 42（Aβ42） in the brains；Caspase-3 and Caspase-9 activity detection kits were applied to detect Caspase-3/9 enzyme activity. Finally，the expression of p-Akt（ser473），total Akt and Caspase-3/9 in the brain tissues were detected by Western blot. Results：Overexpression of Ngb signif－icantly improved the learning and memory and spatial explo－ration ability of the AD mice（P<0.05），and reduced the activities of Caspase-3 and Caspase-9 and the generation of Aβ42 in the hippocampus（P<0.05）. Finally，Western blot results showed that overexpression of Ngb could inhibit the expression of Caspase-3 and Caspase-9 in the hippocampus of the AD mice（P<0.05），and also promote the phosphorylation of p-Akt（ser473）（P<0.05）. While，after the treatment of phosphatidylinositol-3 kinase（PI3K） inhibitor LY294002，the activities of Caspase-3 and Caspase-9 were evidently increased（P<0.05）. Conclusion：Overexpression of Ngb has a significant neuroprotective effect on the AD double transgenic（APPswe/PS1dE9） mice，and the mechanisms may be related to the reduction of generation of Aβ42，and the activation of PI3K/Akt signaling pathway，and then the inhibition of Caspase-dependent apoptosis pathway by Ngb.